Progression of type 2 diabetes mellitus tracks with the failure of pancreatic beta cells to compensate for peripheral insulin resistance. Loss of glucose sensitivity, particularly during the sharp rise in blood glucose that follows a meal, is strongly associated with peripheral tissue damage during diabetes. Yet the molecular mechanisms underlying defects in beta-cell glucose sensing during type 2 diabetes are not well understood. This proposal seeks critical information regarding the regulation of glucokinase, which is the glucose sensing protein in insulin-secreting beta cells. Work in the previous funding period focused on hormonal activation of glucokinase through chemical reaction with nitric oxide. These studies revealed important new connections between defects in glucokinase regulation and a genetic form of human diabetes. Even so, major questions remain concerning the mechanism of glucokinase activation and the impact of diabetes-related cell stress on glucokinase function. Three aims are proposed. Aim 1 will utilize a newly developed glucokinase biosensor to reveal the connection between cellular activation by nitric oxide and the underlying biochemical states suggested by its molecular structure. Aim 2 will focus on understanding the molecular mechanism that leads to glucokinase association with nitric oxide synthase, which is a critical interaction required for cellular activation of glucokinase. Experiments in this aim wil also reveal whether defects in glucokinase regulation may explain the association of NOS1AP gene mutations with human diabetes. Aim 3 will focus on the impact of diabetes-related cell stress on glucokinase activation. Our preliminary data show that impaired endoplasmic reticulum function disrupts glucokinase regulation. The planned studies seek to identify the mechanism behind this disruption, and test whether diet-related obesity can similarly disrupt glucokinase regulation. If so, these studies will provide a molecular explanation for inhibited glucose sensing during type 2 diabetes. In summary, these studies have the potential to unify cellular and biochemical models of glucokinase function, identify new molecular regulators of glucokinase activity, and will lead to new ideas about the molecular causes underlying the deficit in -cell glucose sensing observed in type 2 diabetes mellitus. Understanding the molecular events that worsen type 2 diabetes mellitus is vital for designing new therapies that target beta-cell glucose sensing.